HSV-1, one of nine human herpesviruses (HHVs), has infected more than 3.7 billion people under the age of 50 (67% of the world?s population) and is associated with recurrent cold sores, genital lesions, keratitis, corneal blindness, encephalitis and disseminated neonatal infections. The ability of herpesviruses to establish latent infections that undergo periodic reactivation significantly contributes to their ability to cause life-long disease, viral shedding and transmission to new hosts, underscoring the critical need for a better understanding of their mechanism of DNA replication. Despite the identification of seven viral proteins necessary for HSV DNA synthesis and three origins of replication, the current understanding of the mechanism of viral DNA replication is woefully incomplete. Although it has been known for decades that HSV replicates by concatemer formation, the mechanism by which concatemers form has yet to be identified. Several lines of evidence suggest that HSV promotes a unique form of DNA replication that utilizes a recombination-dependent mechanism to produce concatemers. We have identified a two component HSV recombinase comprised of a 5? to 3? exonuclease (UL12) and a ssDNA annealing protein or SSAP (ICP8) (exo/SSAP). This complex is reminiscent of the ? phage Red ?/? recombinase required for recombination dependent replication in ? phage and in vivo recombination-mediated genetic engineering (recombineering). Interestingly exo/SSAP recombinases are conserved in most dsDNA viruses of bacteria, protozoa, plants and mammals that replicate by concatemer formation. HSV DNA replication is further complicated by the fact that the viral genome contains nicks and gaps, and the introduction this damaged genome is known to activate cellular DNA damage response (DDR) pathways, some of which are antiviral. Thus, HSV has two major challenges: to produce concatemers that can be packaged while at the same time avoiding cellular antiviral processes. ICP8 is a central player in all stages of the DNA replication process, acting as a hub for protein-protein interactions necessary to initiate viral DNA synthesis, bind cooperatively to ssDNA and promote single strand annealing. In Aim 1 we will explore ICP8 interactions required for initiation of DNA replication and cooperative binding to ssDNA. In Aim 2 we will explore the role of ICP8 and UL12 in annealing and recombination-dependent DNA replication. We have shown that the annealing activity of ICP8 is essential for viral DNA replication during infection, supporting the notion that HSV-1 uses recombination-dependent mechanisms during DNA replication. In Aim 3 we will examine the role of UL12 in the manipulation of host DDR pathways. Herpesviruses have evolved an unusual strategy to replicate their genomes that is distinct from bacterial and cellular DNA replication mechanisms. However, the remarkable degree of conservation between viruses that replicate through concatemers underscores the fact that these viruses have evolved an extremely successful strategy utilizing recombination-mediated mechanisms for replication of difficult to replicate genomes.